The generation of distinct apical and basolateral plasma membrane domains is vital to the function of polarized kidney epithelia. In response to ischemia, or as a result of hereditary disease (e.g. polycystic kidney disease or microvillus kidney disease), kidney cells loose their polarity and as a result there is a loss of cellular function and ultimately kidney dysfunction. The cell surface polarity of these cells is achieved by several mechanisms including direct delivery of proteins from the trans-Golgi network, and specialized endocytic pathways that allow endocytosis of plasma membrane proteins/lipids and their subsequent return to the plasma membrane (recycling), selective degradation in lysosomes, or delivery to the opposite plasma membrane domain (transcytosis). Transcytosis is central to the establishment of kidney cell polarity, as it is the only pathway for delivery of newly synthesized membrane proteins to the apical cell surface that is universally found in all epithelial cells examined. Recycling is crucial because it allows these cells to maintain their polarized distribution of proteins and lipids by retrieving both membrane and proteins between adjacent compartments of the endocytotic and biosynthetic pathways. Epithelial cells are known to have distinct populations of apical and basolateral early endosomes, which when labeled with fluid phase markers do not exchange contents. However, it is now known that membrane proteins can transit between basolateral endosomes and a specialized apical endosomal compartment called the apical recycling endosome (ARE). This tubular/vesicular compartment contains not only transcytosing molecules, but recycling ones as well, and is characterized by its accessibility to membrane markers, but inaccessibility to fluid-phase ones. It is hypothesized that this compartment may be a major sorting organelle in polarized epithelia, and may also be the principal site for regulating protein traffic to the apical pole of these cells. The aims of this proposal are to: i. determine if sorting of transcytotic and recycling proteins occurs in the ARE, ii. examine the regulation of proteins recycling basolaterally, and that of proteins recycling and transcytosing from the ARE, and iii. purify the ARE compartment in order to identify other cargo molecules, and proteins that may regulate its function.